恶性疟原虫Pf332抗原的免疫原性分析
发布时间：2019-04-21 17:47
【摘要】： 疟原虫抗药性的产生和扩散已使全球疟疾疫情严重恶化,每年疟疾死亡人数自上世纪七十年代的约50万上升至目前约100万,药物防治疟疾的措施亦面临严重的困难和挑战。因此新的疟疾控制措施的实施已成为当务之急。疫苗接种已使许多种传染病得到有效的控制乃至根除,因此研制有效的疟疾疫苗成为防止疟原虫感染的重要手段。 Pf332是恶性疟原虫基因组所编码的分子量最大的蛋白质。该蛋白质具有典型的跨膜蛋白结构,其膜外区含有一个保守的Duffy抗原结合基团相类似的功能区(Duffy-binding like, DBL domain)。Pf332由红内期恶性疟原虫分泌运输到感染的红细胞表面,在裂殖子侵入红细胞的过程中起到了辅助作用。由于其膜外DBL区与其他膜外区蛋白质相比具有高度的保守性,使其可能成为发展新的抗疟药物和疫苗的候选分子。本研究制备得到12株抗Pf332-DBL区重组蛋白质的IgG1亚型单克隆抗体,通过Western blot和间接免疫荧光实验验证了这12株单抗不仅可以特异性识别重组蛋白质,而且可以识别虫体天然蛋白Pf332。在此基础上,根据Pf332-DBL区氨基酸序列合成9段多肽,以肽扫描的方法确定了单抗的结合肽段,通过单抗体外抑制虫体侵入实验,发现Pf332-DBL区在疟原虫裂殖子侵入红细胞过程中发挥重要作用的两个功能区域位于第1~25个氨基酸和第76~100个氨基酸。在分析Pf332-DBL区重要生物学功能的同时,为确定其在疟疾疫苗研究中的可应用性,本研究还对Pf332-DBL区重组蛋白质的免疫原性进行了分析。通过比较Pf332-DBL区重组蛋白质与4种不同佐剂混合免疫动物后的抗体效价、抗体亚型及抗体维持水平,揭示出Pf332-DBL区具有很强的免疫原性,而且筛选到适合Pf332-DBL区在疫苗研究中的佐剂ISA720。 本研究发现Pf332是疟原虫的一个重要功能蛋白质,DBL区是Pf332蛋白实施生物学功能的关键区域。Pf332-DBL区具有很强的免疫原性,是抗疟原虫的重要疫苗候选抗原。
[Abstract]:The emergence and spread of drug resistance of Plasmodium malaria has seriously worsened the global malaria epidemic. The number of malaria deaths per year has increased from about five hundred thousand in the 1970s to about 1 million at present. The measures to control malaria by drugs are also facing serious difficulties and challenges. Therefore, the implementation of new malaria control measures has become a top priority. Vaccination has resulted in the effective control and eradication of many infectious diseases. Therefore, the development of effective malaria vaccines has become an important means to prevent the infection of Plasmodium falciparum. Pf332 is the largest protein encoded by Plasmodium falciparum genome. The protein has a typical transmembrane protein structure, and its extracellular region contains a conserved Duffy antigen-binding group-like functional area (Duffy-binding like, DBL domain). Pf332) secreted by Plasmodium falciparum in the erythrocytic stage to transport to the surface of infected red blood cells. It plays an auxiliary role in the process of merozoite invading red blood cells. Because of its highly conservative nature compared with other proteins in the extracellular DBL region, it may become a candidate molecule for the development of new antimalarial drugs and vaccines. In this study, 12 monoclonal antibodies against Pf332-DBL domain recombinant protein IgG1 subtype were prepared. The results of Western blot and indirect immunofluorescence test showed that these 12 monoclonal antibodies not only specifically recognized the recombinant protein, but also showed that these monoclonal antibodies could specifically recognize the recombinant protein. And it can recognize the natural protein Pf332.. On the basis of this, nine peptides were synthesized according to the amino acid sequence of Pf332-DBL region. The binding peptides of monoclonal antibodies were determined by peptide scanning, and the in vitro inhibition of insect invasion by monoclonal antibodies was carried out. It was found that the two functional regions which play an important role in the invasion of erythrocytes by merozoites of Plasmodium falciparum are located at 1 ~ 25 amino acids and 76 ~ 100 amino acids. In order to determine the applicability of Pf332-DBL region in malaria vaccine research, the immunogenicity of recombinant protein in Pf332-DBL region was also analyzed in this study. By comparing the antibody titer, antibody subtype and antibody maintenance level between the recombinant protein of Pf332-DBL region and four different adjuvants, it was revealed that the Pf332-DBL region had a strong immunogenicity. And the adjuvant ISA720., which is suitable for Pf332-DBL region in vaccine research, was screened out. In this study, we found that Pf332 is an important functional protein of Plasmodium malaria, and DBL region is the key region of the biological function of Pf332 protein. Pf332-DBL region has strong immunogenicity and is an important vaccine candidate antigen of Plasmodium malaria.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392
本文编号：2462421
[Abstract]:The emergence and spread of drug resistance of Plasmodium malaria has seriously worsened the global malaria epidemic. The number of malaria deaths per year has increased from about five hundred thousand in the 1970s to about 1 million at present. The measures to control malaria by drugs are also facing serious difficulties and challenges. Therefore, the implementation of new malaria control measures has become a top priority. Vaccination has resulted in the effective control and eradication of many infectious diseases. Therefore, the development of effective malaria vaccines has become an important means to prevent the infection of Plasmodium falciparum. Pf332 is the largest protein encoded by Plasmodium falciparum genome. The protein has a typical transmembrane protein structure, and its extracellular region contains a conserved Duffy antigen-binding group-like functional area (Duffy-binding like, DBL domain). Pf332) secreted by Plasmodium falciparum in the erythrocytic stage to transport to the surface of infected red blood cells. It plays an auxiliary role in the process of merozoite invading red blood cells. Because of its highly conservative nature compared with other proteins in the extracellular DBL region, it may become a candidate molecule for the development of new antimalarial drugs and vaccines. In this study, 12 monoclonal antibodies against Pf332-DBL domain recombinant protein IgG1 subtype were prepared. The results of Western blot and indirect immunofluorescence test showed that these 12 monoclonal antibodies not only specifically recognized the recombinant protein, but also showed that these monoclonal antibodies could specifically recognize the recombinant protein. And it can recognize the natural protein Pf332.. On the basis of this, nine peptides were synthesized according to the amino acid sequence of Pf332-DBL region. The binding peptides of monoclonal antibodies were determined by peptide scanning, and the in vitro inhibition of insect invasion by monoclonal antibodies was carried out. It was found that the two functional regions which play an important role in the invasion of erythrocytes by merozoites of Plasmodium falciparum are located at 1 ~ 25 amino acids and 76 ~ 100 amino acids. In order to determine the applicability of Pf332-DBL region in malaria vaccine research, the immunogenicity of recombinant protein in Pf332-DBL region was also analyzed in this study. By comparing the antibody titer, antibody subtype and antibody maintenance level between the recombinant protein of Pf332-DBL region and four different adjuvants, it was revealed that the Pf332-DBL region had a strong immunogenicity. And the adjuvant ISA720., which is suitable for Pf332-DBL region in vaccine research, was screened out. In this study, we found that Pf332 is an important functional protein of Plasmodium malaria, and DBL region is the key region of the biological function of Pf332 protein. Pf332-DBL region has strong immunogenicity and is an important vaccine candidate antigen of Plasmodium malaria.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392
【引证文献】
相关期刊论文 前1条
1 文雪霞;陈化兰;熊永忠;王秀荣;;抗原表位鉴定方法的研究进展[J];中国畜牧兽医;2012年07期
相关硕士学位论文 前1条
1 武闯;日本血吸虫23kDa膜蛋白与人免疫球蛋白的相互作用分析[D];吉林大学;2011年
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